Microbial EcologyAdapted from Environmental Molecular Microbiology and Metagenomics: Theory, Methods and Applications
Microbial Diversity and PhylogenyThe small subunit ribosomal RNA gene (SSU rRNA) has been the cornerstone of microbial ecology studies over the last 15 years, and has provided much of what we know about Bacterial and Archaeal diversity and community structure, and has greatly aided microbial taxonomy. Genomics is aiding our understanding of the relationships among closely related organisms, and ultimately of natural populations.
In a recent study the available 16S rRNA genes from species type strains were examined. The most distant sequences in the median genus and family were about 4.4% and 14% different, respectively. The largest dissimilarity between a sequence and its closest relative in the same taxa (similar to single-linkage clustering distance) was 3.5% and 10% for the median genus and family. The ratio of the two values averaged less than 1.5 for all ranks, indicating that most taxa are not elongated, but are fairly spherical. When the near-full-length 16S rRNA gene sequences in the public databases were clustered into groups at proxy distances for species, genus, family and order, the number of clusters with time increased exponentially for all ranks documenting the enormous diversity of the microbial world. (Recommended reading: The Archaea)
Genomics and MetagenomicsThe extensive suite of molecular-based approaches developed over the past decade has enabled the field of metagenomics, the study of uncultured microorganisms. Paramont to metagenomic analysis is use of high-throughput DNA sequencing technologies, which with the advent of low cost next-generation methods is transforming metagenomics. The application of metagenomics, to both global environments and microbes associated with a living host, has facilitated study of the functional ecology of environmental microorganisms. Novel functional genes and environmental functional signatures can be retrieved using metagenomics, and these can form the basis of hypothesis driven analyses of uncultured microorganisms. A with any technology, the daunting task is to understand and apply the growing number of metagenomic sequences in the context of microbial ecology and evolution.
MetaproteomicsMicrobial ecology is currently experiencing a renaissance spurred by the rapid development of molecular techniques and "omics" technologies in particular. As never before, these tools have allowed researchers in the field to produce a massive amount of information through in situ measurements and analysis of natural microbial communities, both vital approaches to the goal of unraveling the interactions of microbes with their environment and with one another. While genomics can provide information regarding the genetic potential of microbes, proteomics characterizes the primary end-stage product, proteins, thus conveying functional information concerning microbial activity. Advances in mass spectrometry instrumentation and methodologies, along with bioinformatics approaches, have brought this analytic chemistry technique to relevance in the biological realm due to its powerful applications in proteomics. Mass spectrometry-enabled proteomics, including "bottom-up" and "top-down" approaches, is capable of supplying a wealth of biologically-relevant information, from simple protein cataloging of the proteome of a microbial community to identifying post-translational modifications of individual proteins.
Nucleic-Acid-based CharacterizationNucleic acid-based techniques were first used to characterise natural microbial communities in the early 1990s and are now used routinely. The ability to characterise communities without the requirement for cultivation has led to enormous advances in our ability to describe microbial communities and to determine the factors that influence their structure. New generations of molecular techniques provide even greater descriptive power and can be used to assess the physiological potential and ecosystem function of communities. They also enable microbial ecologists to address fundamental questions in population and community ecology, including investigation of the links between diversity and function. This chapter describes methods currently used to analyse nucleic acids extracted from environmental samples, and shows how they can be used to characterise communities. It also looks ahead to exciting new technologies that are likely to increase greatly our ability to explore and understand the complex functions and interactions of microbial communities in natural environments.
Microarrays in Microbial EcologyMicroarrays have proven to be a useful and high-throughput method to provide targeted DNA sequence information for up to many thousands of specific genetic regions in a single test. A microarray consists of multiple DNA oligonucleotide probes that, under high stringency conditions, hybridize only to specific complementary nucleic acid sequences (targets). A fluorescent signal indicates the presence and, in many cases, the abundance of genetic regions of interest. In this chapter we will look at how microarrays are used in microbial ecology, especially with the recent increase in microbial community DNA sequence data. Of particular interest to microbial ecologists, phylogenetic microarrays are used for the analysis of phylotypes in a community and functional gene arrays are used for the analysis of functional genes, and, by inference, phylotypes in environmental samples. A phylogenetic microarray that has been developed by the Andersen laboratory, the PhyloChip, will be discussed as an example of a microarray that targets the known diversity within the 16S rRNA gene to determine microbial community composition. Using multiple, confirmatory probes to increase the confidence of detection and a mismatch probe for every perfect match probe to minimize the effect of cross-hybridization by non-target regions, the PhyloChip is able to simultaneously identify any of thousands of taxa present in an environmental sample. The PhyloChip is shown to reveal greater diversity within a community than rRNA gene sequencing due to the placement of the entire gene product on the microarray compared with the analysis of up to thousands of individual molecules by traditional sequencing methods. A functional gene array that has been developed by the Zhou laboratory, the GeoChip, will be discussed as an example of a microarray that dynamically identifies functional activities of multiple members within a community. The recent version of GeoChip contains more than 24,000 50mer oligonucleotide probes and covers more than 10,000 gene sequences in 150 gene categories involved in carbon, nitrogen, sulfur, and phosphorus cycling, metal resistance and reduction, and organic contaminant degradation. GeoChip can be used as a generic tool for microbial community analysis, and also link microbial community structure to ecosystem functioning. Examples of the application of both arrays in different environmental samples will be described in the two subsequent sections.
The Soil EnvironmentKornelia Smalla and Jan Dirk van Elsas Until fairly recently, the living soil has been considered as a functional black box that is intrinsically too difficult to be unravelled into its core components. However, this concept has changed with the advent of the modern methodologies. The intricacies of microbial life in soil has been impacted by the advanced, mainly molecularly-based, approaches that have been unleashed on the soil habitat in recent years. The application of molecular and other advanced methods (cultivation-independent analyses) has provided exciting new insights into microbial life in soil.
Soil is an extremely diverse and complex habitat containing many microsites and gradients that form a range of different biogeochemical interfaces. Depending on the proportion of sand, silt and clay, the surface area in soil can vary from 11 cm2 up to 8 million cm2 per gram of soil read more.... The aggregates formed by minerals, soil organic matter, fungal hyphae, roots and plant debris offer a range of potential niches for microorganisms with different lifestyles. The architecture of the soil pore network essentially defines the habitat colonized by the microorganisms and the pore space strongly influences the nature and extent of the interactions between the organisms inhabiting the soil. The heterogeneous physical structure of soil affects the spatial distribution of water, oxygen and nutrients, which in turn influences the composition and activity of the microbial communities themselves. As an example, the spatial distribution of bacteria in topsoil and subsoil was found to be different, but lateral variations in spatial distributions are also likely to occur.
In terms of their occurrence in microsites, bacteria can be found in soil as single cells but most often they occur as microcolonies, i.e. small agglomerates of cells that can be regarded as primative soil biofilms read more.... Microorganisms are the major drivers of geochemical and biotransformation processes in soil. In concert with the soil's inorganic and organic constituents, microbes are influential in actively shaping the architecture of the soil matrix by the formation and restructuring of soil aggregates. In addition, the diversity of microbial communities is extremely high in most soils. There are only a few quantitative estimates of the numbers of microbial taxa that can co-exist in just a single gram of soil, but an advanced analysis of nucleic acid-based analyses, based on re-association kinetics, has suggested that prokaryotic diversity can reach 1 million species genomes per g, which by far exceeds the common estimates of bacterial richness in soil obtained from cultivation-based studies read more....
A major driving force that spurs the microbial diversity of soil is the enormous heterogeneity of the soil habitat, allowing the formation of numerous niches. Different factors, such as the presence or absence of water, soil pH, temperature, redox potential and the soil organic matter content do not only influence the types of microbes colonizing the respective microniche but also their activity. All these factors can vary greatly between the different microhabitats and, thus, not only the composition but also the activity and interactions of the microbiota will largely vary due to the spatial and temporal heterogeneity between as well as within the microsites.
A key determinant of microbial fitness in soil is the ability of microbial cells to fine-tune their cellular metabolism to the abiotic and biotic conditions that prevail locally. In addition, the rate of adaptation of microorganisms to changing environmental conditions might be enhanced by horizontal gene transfer processes read more.... Undoubtedly, the most important prerequisite for microbial life in soil is the availability of water. Next to being indispensable for microbial life, the water in soil carries dissolved gases, ions and nutrients to microorganisms, and, in cases of saturation, may quickly establish anaerobic conditions. For instance, an increase of the moisture content of soil can greatly influence the microbial communities that are locally present, in particular by connecting pore spaces in and among aggregates that were unconnected without water, thus increasing the aggregate connectivity. Predation by protozoa or Bdellovibrio species will therefore be particularly enhanced in relatively wet soils.
Plant Microbial CommunitiesPlants, both above- and below ground, offer diverse habitats for microbial colonization and growth. Plant-microbe interactions lie at the heart of plant performance and ecology. Plants provide various growth substrates and physical habitats for microbes on both sides of the air-soil interface, and numerous plant-associated niches have been exploited by specific microbial species, either by specializing on the distinct environmental conditions available, or entering into commensal, mutualistic, or parasitic interactions with plants. This chapter seeks to examine the state of the art with respect to our ability to characterize the structure, function and interactions of plant-associated microbial communities, with a particular focus on the role of molecular biological methods and environmental genomics strategies in promoting this field. We will pay particular attention to bacterial and fungal colonization of above and belowground plant surfaces (phyllosphere and rhizosphere, respectively), as well as in planta (endosphere) interactions of endophytic, parasitic and symbiotic microorganisms. Of particular importance to advancing this research field are emerging methodologies, including novel '-omics' approaches, that seek to link microbial identity to in situ functioning, and holistic approaches that capture the complexities involved in multiple plant-microbe interactions. (Recommended reading: Plant Pathogenic Bacteria)
Marine Microbial EnvironmentsOcean microbial communities play important roles in global geochemical cycles. From the earliest cultivation experiments to today's metagenomic analyses, most of the major discoveries in this field were driven by applications of novel methods. Molecular ecology had a major impact by revealing the true scope of microbial diversity and providing genetic markers that could be used to track important species, even in cases where cultures were unavailable. In some cases, metagenomics provided insight into the biochemical adaptations of these organisms. A renaissance in culturing technique led to isolates of many abundant ocean microbes that could then be studied in a laboratory setting. Today a consortium of approaches that span scales from molecules to ocean basins are being applied to ocean micobial ecosystems, with the result that marine microbiology is becoming a highly integrated science.
Human Microbial EnvironmentApplications of recent advances in molecular methods have illuminated the previously hidden diversity of the microbial world that not only inhabits our bodies, but that also lives in a close symbiotic association with us. This human-associated microbiota, or human microbiome, is responsible for many key functions in our bodies. Increasing evidence suggests many important roles of individual members of the human microbiome and their respective influences towards ultimate health and disease of the host. This chapter highlights some of the important functions of the human microbiome, many of which were gleaned using different molecular approaches. The clinical field has thus greatly benefited from the molecular toolbox that was initially developed by microbial ecologists for investigation of other complex ecosystems, such as soil. As the field has progressively moved away from a dependence on cultivation-based approaches towards increasing reliance on molecular approaches, the amount of knowledge about the human microbiome composition and function has greatly expanded. Most recent studies using molecular tools, including various 'omics' approaches, have focused on the intestinal microbiota. Therefore, we have also primarily discussed the gut microbiota in this chapter. In addition, the influence of difference host-related factors, such as genetics, age, birth mode, diet and geographical location are discussed with respect to their impact on the composition and related function of the human microbiome. Some beneficial bacteria, such as probiotic strains, are beneficial whereas others are detrimental to human health. Some of the latter include correlations of microbial compositions to intestinal diseases and cancer. The more information that we have about the key roles of specific members of the human microbiome, the more potential we have for manipulation of the composition of the microbiota to enhance the prevalence of beneficial species and to diminish the amounts of detrimental ones. This is a guiding vision for future research in this area. (Recommended reading: Lactobacillus and Probiotics and Prebiotics)
Wastewater TreatmentSatoshi Okabe and Yoichi Kamagata Of Earth's diverse microbial habitats, wastewater treatment processes are one of the most elaborate anthropogenic niches geared towards one purpose: cleaning up water. Recent application of molecular techniques is unveiling the microbial composition and architecture of the complex communities involved in the treatment processes. It is now recognized that wastewater processes harbor a vast variety of microorganisms most of which are yet-to-be cultured, hence uncharacterized. In this chapter, the latest knowledge on diversity, structure and functions of microbial communities in nitrifying processes, anaerobic ammonia oxidation processes and methane fermenting processes are summarized.
Bacterial BiofilmsMany bacteria can grow and live as biofilms, in which single microbial cells individually interconnect with each other through an extracellular matrix. Biofilm-forming bacteria pose severe problems in the environment, industry and health care sector due to increased bacterial survival competence in the environment and the protective nature of biofilms that prevent effective eradication. Technological progress in microscopy, molecular genetics and genome analysis has significantly advanced our understanding of the structural and molecular aspects of biofilms, especially of extensively studied model organisms such as Pseudomonas aeruginosa. Biofilm development can be divided into several key steps including attachment, microcolony formation, biofilm maturation and dispersion; and in each step bacteria may recruit different components and molecules including flagella, type IV pili, DNA and exopolysaccharides. The rapid progress in biofilm research has also unveiled several genetic regulation mechanisms implicated in biofilm regulation such as quorum sensing and the novel secondary messenger cyclic-di-GMP. Understanding the molecular mechanisms of biofilm formation has facilitated the exploration of novel strategies to control bacterial biofilms.
- Environmental Molecular Microbiology
- Metagenomics: Theory, Methods and Applications
- Environmental Microbiology
- MALDI-TOF Mass Spectrometry in Microbiology
- Climate Change and Microbial Ecology: Current Research and Future Trends
- Gas Plasma Sterilization in Microbiology: Theory, Applications, Pitfalls and New Perspectives
- Flow Cytometry in Microbiology: Technology and Applications